Hepes

Roflumilast N-oxide (RNO) was provided by Nycomed (Konstanz, Germany). F-12K Nutrient Mixture Kaighn's Modification cell culture medium, antibiotics, glutamine and trypsin-EDTA were purchased from Invitrogen (Eugene, OR, USA). Fetal calf serum (FCS) was from Hyclone (Logan, UT, USA). Lipolysaccharide from E. coli 055:B5, Thiazolyl Blue Tetrazolium Blue (MTT), SB-203580, SP-600125 and U0126 were purchased from Sigma-Aldrich (St. Louis, MO, USA). The specific antibodies against phospho-(p44/42) ERK1/2, (p44/42) ERK1/2, phospho-p38 MAP kinase, p38 MAP kinase, phospho-SAPK/JNK, SAPK/JNK, were purchased from Cell Signaling Technology (Beverly, MA, USA). Acrylamide, SDS, Tris, HEPES and BSA were purchased from Eurobio (Les Ulis, France). Bradford protein assay and precision plus protein dual color standards were purchased from Bio-Rad (Hercules, CA, USA).

The T. cruzi soluble Ags (TcSAs) were obtained using previously described methods, with some modifications (44 (link)–46 (link)). Briefly, T. cruzi trypomastigotes (Y strain) were obtained on a monolayer of VERO cells (44 (link), 45 (link)), which were cultured in DMEM (Eurobio, Les Ulis, France) supplemented with 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, and 0.01 M HEPES (Eurobio; Les Ulis, France) at 37°C in a humidified atmosphere of 5% CO₂. Amastigotes and trypomastigotes (1:1 ratio) were collected from the VERO cell culture supernatants at 96–120 h post-infection (44 (link)). Then, the parasites were washed twice with cold 1 × PBS (Eurobio) and resuspended at a density of 1 × 10⁶ parasites/μl in lysis buffer as previously reported (46 (link)). Parasites were incubated on ice for 30 min, and the supernatants containing TcSAs were collected by centrifugation at 12,000 × g for 15 min at 4°C and stored at −80°C until use. The protein concentrations were determined using the Bradford assay, and the protein profiles were analyzed using SDS-PAGE followed by Coomassie blue staining (Gibco BRL; Grand Island, NY, USA).

One million of epimastigote forms of T. cruzi MHOM/BR/00/Y (DTU II) [63 ] were grown in Liver Infusion Tryptose (LIT) medium supplemented with 10% bovine serum albumin (Eurobio, Les Ulis, France) at 26 °C; after 96 h of culture, 2 × 10⁸ epimastigotes were obtained. In the meantime, Green Monkey renal fibroblast-like cells (Vero cells; ATCC CCL-81, Manassas, VA, USA) were grown in Dulbecco’s modified eagle medium (DMEM) (Eurobio, Les Ulis, France) supplemented with 10% bovine serum albumin (Eurobio, Les Ulis, France), 2 mM L-glutamine, 100 U/mL penicillin, 100 mg/mL streptomycin, and 0.01 M hepes (Eurobio, Les Ulis, France) at 37 °C in a humid atmosphere with 5% CO₂. After reaching semiconfluence, the Vero cells were incubated for 10 h with 3 × 10⁷T. cruzi trypomastigotes MHOM/BR/00/Y (DTU II) [63 ], and the parasites were recovered at 96 h postinfection. Trypomastigotes with three cyclic passages in Vero cells were used in subcellular location assays. Similarly, 10⁶ promastigotes of Leishmania major MHOM/IL/81/Friedlin strain [64 (link)] were grown in Schneider’s insect medium (Sigma-Aldrich, St. Louis, MO, USA), supplemented with 20% bovine serum albumin (Eurobio, Les Ulis, France), and 1 μg/mL of 6-biopterin (Sigma-Aldrich, St. Louis, MO, USA) at 26 °C; after 72 h of culture 8 × 10⁶ promastigotes in logarithmic growth phase were obtained.